Tissue protein structures such as collagen and actomyosin give rise to intense second harmonic generation (SHG), and the resulting contrast yields high-resolution images of live tissues on a laser-scanning SHG microscope. The physics describing SHG indicates that information on secondary and higher-order protein structure may be deduced from the interactions of these proteins with specific polarizations of light. Miniaturized technology to acquire laser-scanning images is developing quickly. Therefore, if warranted, a dedicated engineering effort could soon permit physicians to analyze microscopic and submicroscopic structure in muscle and connective tissues of live patients. However, the key questions now are: i) whether medically valuable information can be derived from SHG images, ii) which tissues and medical conditions are most readily analyzed, iii) what the most optimal and simplest means will be for collecting SHG images in each tissue, iv) which of the physical properties of SHG might lead to medically-important insights into native protein structure, and v) whether the optics required for fine structural analysis will apply in the context of live tissues. These questions define two basic paths. First is characterization of SHG image profiles from various tissues (including developmental and diseased states), and optimization of parameters for generating SHG contrast images from each protein/tissue type. Second is analysis of the molecular and physical basis of SHG from tissue protein arrays, and testing of the predicted power of SHG polarization analysis to probe sub-microscopic structure of protein assemblies in tissues. Our specific aims follow each of these promising paths, and will seek convergence between the two. These aims are i) Test the utility of SHG imaging as a diagnostic modality to reveal histology/pathology of specific tissues: skeletal and cardiac muscle, skin, cartilage, bone, and plaque deposits caused by disease. ii) Use detailed polarization analysis and super-resolution techniques to characterize the molecular sources of SHG in tissues, the structural states of these molecules that underlie this optical activity, and the response of their optical properties to changes in molecular structure. iii) Determine ideal optical parameters for SHG imaging of tissues in vivo.